Innate immune responses combat infectious microbes by driving inflammation, host-defense pathways and adaptive immunity. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional on and off switches that account for the specificity of gene expression in response to microbial triggers. Well-defined DNA binding transcription factors, transcriptional co-regulators and chromatin modifying complexes collaborate to coordinate transcription of immune genes. Recent evidence indicates that non-coding RNAs also contribute to gene regulation in diverse biological contexts. A growing body of evidence including our own published studies supports their importance in the immune system. lncRNAs have been identified in macrophages, dendritic cells, neutrophils, T-cells and B-cells during their differentiation and/or activation. We have found that these lncRNAs in turn regulate expression of other immune genes. This proposal is based on our hypothesis that lncRNAs are associated with chromatin in immune cells in order to regulate transcription of immune genes. We propose to identify and catalogue chromatin-associated lncRNAs in both resting and activated human macrophages and employ cutting edge molecular approaches to functionally characterize the genomic targets of these regulators. These objectives will be met using two specific aims: In aim 1 we will identify chromatin-associated lncRNAs (ca-lncRNAs) in an unbiased manner in human macrophages using sucrose density gradient fractionation as well as chromatin Immunoprecipitation coupled to RNA-sequencing. The regulation of these ca-lncRNAs will then be profiled using Nanostring technology to assess the temporal regulation and signaling mechanisms regulating their expression in response to microbial products, viral and bacterial pathogens. In aim2 we will identify the genomic targets and protein-binding partners of chromatin-associated lncRNAs using RNA antisense purification (RAP) coupled to DNA-Sequencing or Mass Spectrometry, respectively. These studies will define functionally important lncRNAs acting at the level of chromatin to modulate macrophage transcriptional responses and define genomic targets of ca-lncRNAs regulated during host-pathogen interactions. This unbiased identification of functionally relevant lncRNAs will also provide a platform for future studies that characterize these genes in greater detail. They will also provide new strategies to study lncRNA in other biological contexts.